Four Dictyostelium myosin I isoforms have been characterized. DMIB and DMID have tail domains that are predicted from sequence to interact with both membranes and F-actin, are strongly upregulated during chemotactic aggregation, and are localized to the leading edge of migrating cells, the polar ruffles of dividing cells, sites of phagocytosis, and sites of cell:cell contact. The DMIA and DMIE isoforms have tail domains predicted to interact with membranes only, are not upregulated during chemotactic aggregation, and do not colocalize with DMIB and DMID (preliminary studies with alphaDMIA antibody show punctate staining of the cytoplasm). Together, these results are consistent with the existence of at least two subfamilies of myosin I in Dictyostelium which may subserve different functions in the cell (with DMIB/DMID being primarily involved in cell shape change and DMIA/DMIE in intracellular membrane organization and movement). With regard to generating myosin I mutants and characterizing their phenotype, we are currently analyzing three mutants: DMIB-, DMID- and DMIB-/DMID antisense cells. DMIB- cells show reduced efficiencies of chemotactic aggregation, phagocytosis, and morphogenesis. Furthermore, analysis of DMIB- cells by quantitative video microscopy has revealed clear cut abnormalities in cell shape change and locomotion. Similar analyses of the DMIB-/DMID antisense double mutants reveal, however, that the additive effect of suppressing the expression of DMID by >99% in DMIB- cells using antisense RNA is minimal. Therefore, a third member of the DMIB/DMID subfamily (DMIC) is being targeted for disruption in the double mutant. A high-molecular-weight form of Acanthamoeba myosin I has been partially purified by a combination of chromatographic techniques. Efforts to complete the purification, including removal of contaminating myosin II using an antibody column, are underway. A possible homolog of the dilute/p190/myo2 class of nonmuscle myosin (two headed but nonflamentous) has been cloned from Dictyostelium by PCR. The PCR product hybridizes to an approximately 5.8 kDa mRNA and has been used to obtain genomic clones which are currently being analyzed. A full- length clone for mouse dilute has been constructed for use in future studies with cultured neurons and melanocytes.